Female mules are considered as infertile; however, they could be used as recipients in interspecific embryo transfer. This study reports for the first time how it is possible to obtain the birth of a live Andalusian donkey foal after transfer a donkey embryo to a non-cycling mule. Two non-cycling mules were used as recipients, oestradiol benzoate was administered when donors showed oestrus and long-acting progesterone after ovulation. The mules also received long-acting progesterone every 7 days until 120 days of gestation. One embryo was collected from the two donor jennies and transferred to one of the mules after 5 days of progesterone treatment. Pregnancy was established and maintained after embryo transfer. The pregnant mule carried to term and delivered a live donkey foal after 375 days of pregnancy. In conclusion, non-cycling mules treated with oestradiol benzoate and long-acting progesterone can be successfully used as recipients of donkey embryos, which open new ways for the conservation of endangered donkey species.

Authors' contributions: Contributed to the study execution: MB and TF. Drafting the manuscript: IO and MH. Conceived and designed the study and wrote the paper: JD. All authors read and approved the final manuscript.

The population of the Andalusian donkey is estimated at 793 individuals (DAD-IS-FAO, 2014), therefore it is considered as endangered (BOE, 2009). Equine embryo transfer (ET) permits to obtain several foals from a mare each year (Stout, 2006), becoming a valuable strategy for preserving endangered equid species (Summers et al., 1987). However, studies concerning ET in endangered donkeys are scarce and discouraging results have been obtained (Camillo et al., 2010; Panzani et al., 2012). Recipient's pregnancy rate depends on the quality of recipients and their synchronization with donors (Carnevale et al., 2000; Squires et al., 2003). This implies needing a significant number of recipients for each donor, which is difficult to conduct in endangered species. Interspecific ET and the use of non-cycling recipients could be a successful long-term strategy to increase the number of recipients in ET programs.

The study was started in the Centro Rural Malpica (Palma del Río, Cordoba, Spain) in May 2015. All animal procedures were performed in accordance with the Spanish laws for animal welfare and experimentation (BOE, 2013).

Two healthy mules (4-years-old and in good body condition) in anoestrus were used as embryo recipients. When the donor jenny was mated the first time, the mule recipients received two intramuscular injections of 5 mg oestradiol benzoate (Sincrodiol®, Ourofino Saûde Animal, Cravinhos, Brazil) at an interval of 24 h. After the donor jenny ovulated, recipients were given 1500 mg of long-acting progesterone (P4-LA/IM, Brazil) intramuscularly every 7 days until day 120 of pregnancy (Day 0 = day of ET).

Embryo collections were done on day 7 after ovulation (Camillo et al., 2010). Embryos were evaluated under stereosmicroscope, washed 10 times in Syngro® holding (Bioniche Animal Health, Washington, USA) and loaded into 0.25 ml straws until transferred transcervically to the mule recipients.

Pregnancy diagnosis was performed on days 5 and 25 after ET by ultrasound. Pregnancy was monitored weekly until day 120 after ET and thereafter monthly until parturition. Gender determination was also performed at day 120 (Bucca, 2005).

Figure 1.
Ultrasonography images (5.0 MHz) of the conceptus
from the non-cycling mule recipient at different
gestational days: (a) Embryonic vesicle consisting of a
yolk sac (YS) at 12 days of gestation (D 0 = ovulation).
The shape of the embryonic vesicle was spherical and the
diameter was 1.3 mm. (b) Embryo proper (EP) aging 25
days in the embryonic vesicle. The shape of the vesicles
was irregular and the diameter was 48 × 28 mm. (c) Fetus
(F) was in the ventral pole at day 40 and the allantoic sac
(AS) is observed. The shape of the vesicles was irregular
and the diameter was 56 × 20 mm. (d) At day 120, male
foetal gonads (MFG) have a homogeneous texture with a
thin central longitudinal echogenic lined vessel.

Only one embryo (morula stage grade 1 with a diameter of 175 µm) was recovered and transferred to one of the non-cycling recipient mules at day 5 of progesterone treatment. The ET resulted in an ongoing pregnancy. Ultrasonography images of the conceptus at different gestational days are shown in Figure 1. Foetal viability was confirmed by ultrasound each month of pregnancy.

Parturition occurred spontaneously on day 375 of gestation and a live donkey foal was delivered without the need for assistance. The surrogate mule mother expelled the foetal membranes after birth, and presented strong protective behaviour and large amount of milk in the well-developed udders. Gross examination of the foetal membranes did not reveal any lesions of clinical importance; however, the donkey foal had a congenital osteodystrophy with facial hyperostosis. Similar findings were described by Bryant et al. (2012) in a Thoroughbred filly which was born with congenital aneurysmal bone cyst. Although the pathogenesis of these lesions remains unclear, possible causes are malformation of intraosseous vasculature secondary to trauma, bleeding disorders, fibrous dysplasia, hematomas or underlying neoplasia.

This is the first report of a foal born following transfer of a fresh donkey embryo into a non-cycling mule. Some authors have previously used cycling mule as donkey embryos recipients and obtained pregnancies and/or live donkey foals (Silva et al., 2013; González et al., 2015). Likewise, successful non-surgical transfer of horse embryos into cycling mules has been reported (Camillo et al., 2003), but failed to obtain pregnancies when non-cycling mules were used. These unsatisfactory results have been explained by the lower quality of the endometrium in permanently non-cyclic recipients (Camillo et al., 2003) and the lower fertility rates that have this recipients (Carnevale et al., 2000). The hormonal treatment used in this study showed to be effective for preparing anovulatory mares in deep anoestrus and transitional phase as embryo recipients (Rocha Filho et al., 2004; Botelho et al., 2015). Based on our results, the administration of this treatment to non-cyclic mules would be efficient for establishing and maintaining pregnancy after ET.

The eutocic birth of a live donkey foal suggests a normal development of the placenta (Gonzalez et al., 2015). Unfortunately, the foal died few hours after the birth due to right-side heart failure, possibly related to asphyxia. In the previous case reported in literature (Bryant et al., 2012) the filly was euthanized, therefore the relationship between the facial lesions and the spontaneous death of the donkey foal cannot be ensured. We could not confirm the excellent maternal ability of mules reported previously (Camillo et al., 2003; González et al., 2015), but a strong protective behaviour and well-developed udders were observed.

We conclude that non-cycling mules treated with oestradiol benzoate and long-acting progesterone can establish and maintain pregnancy after non-surgical transfer of a 7-day donkey embryo. The pregnant mule can successfully carry the donkey conceptus to term and give birth to a mature donkey foal. If this result would be confirmed in further studies using more animals, the use of non-cycling mules in interspecific ET programs could be a good opportunity for the conservation of endangered donkey breeds.

BOE, 2009. Royal decree 2129/2008, of 26 December, that establish the national program for conservation, improvement and promotion of livestock breeds. Boletín Oficial del Estado [Spain] No. 23, 27/01/09.

BOE, 2013. Royal decree 53/2013, of 1 February, that establish the basic rules to protect animals used in experiments and for other scientific purposes, including teaching. Boletín Oficial del Estado [Spain] No. 34, 08/02/13.